U.S. patent application number 11/602755 was filed with the patent office on 2008-05-22 for methods for amplifying the raman signal of surface enhanced raman scattering nanoparticles.
Invention is credited to Michael Burrell, Frank John Mondello, Tracy Lynn Paxon.
Application Number | 20080118986 11/602755 |
Document ID | / |
Family ID | 39417406 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080118986 |
Kind Code |
A1 |
Burrell; Michael ; et
al. |
May 22, 2008 |
Methods for amplifying the Raman signal of surface enhanced Raman
scattering nanoparticles
Abstract
Methods for amplifying the Raman signal of primary SERS
nanoparticles are provided. One method generally includes binding
secondary SERS particles to the primary SERS nanoparticles after
binding of the primary SERS nanoparticles. In another method,
secondary SERS nanoparticles are brought in close proximity to the
primary SERS nanoparticles, wherein the secondary nanoparticles are
free of a reporter molecule or have a reporter molecule different
from that of the primary SERS nanoparticles.
Inventors: |
Burrell; Michael; (Clifton
Park, NY) ; Mondello; Frank John; (Niskayuna, NY)
; Paxon; Tracy Lynn; (Waterford, NY) |
Correspondence
Address: |
GENERAL ELECTRIC COMPANY;GLOBAL RESEARCH
PATENT DOCKET RM. BLDG. K1-4A59
NISKAYUNA
NY
12309
US
|
Family ID: |
39417406 |
Appl. No.: |
11/602755 |
Filed: |
November 21, 2006 |
Current U.S.
Class: |
436/166 |
Current CPC
Class: |
G01N 33/587 20130101;
B82Y 15/00 20130101; G01N 21/658 20130101 |
Class at
Publication: |
436/166 |
International
Class: |
G01N 21/31 20060101
G01N021/31 |
Claims
1. A process for amplifying a Raman signal associated with primary
SERS nanoparticles, wherein the SERS nanoparticles contain a
metallic core, the process comprising: aggregating secondary SERS
nanoparticles about the primary SERS nanoparticles after a binding
event between the primary SERS nanoparticle and a targeted analyte,
wherein the secondary SERS nanoparticles contain a different
reporter molecule than the primary SERS nanoparticles or are free
from the reporter molecule.
2. The process of claim 1, wherein the primary SERS nanoparticles
have a glass coating of less than 5 nanometers.
3. The process of claim 1, wherein the binding event comprises
binding at least one of the primary SERS nanoparticles to a target
organism and amplifying the Raman signal associated with the at
least one of the primary SERS nanoparticles.
4. The process of claim 1, wherein the metal core contains a metal
selected from a group consisting of gold, silver, copper, sodium,
aluminum, chromium, and alloys containing at least one of these
metals.
5. The process of claim 1, wherein the primary SERS nanoparticles
have a negative charge and the secondary SERS nanoparticles have a
positive charge.
6. The process of claim 1, wherein the primary SERS nanoparticles
have a positive charge and the secondary SERS nanoparticles have a
negative charge.
7. The process of claim 1, wherein said aggregating secondary SERS
nanoparticles comprises depositing a solution containing the
secondary SERS nanoparticles onto the primary SERS nanoparticles;
and precipitating the secondary SERS nanoparticles by drying the
solution.
8. The process of claim 1, wherein said aggregating secondary SERS
nanoparticles comprises destabilizing a solution of the secondary
SERS nanoparticles by addition of a destabilizing material and
forcing the solution containing the secondary SERS nanoparticles to
aggregate about the primary SERS particles.
9. The process of claim 8, wherein said destabilizing a solution of
the secondary SERS nanoparticles comprises precipitating a portion
of the secondary SERS nanoparticles and forming aggregates about
the primary SERS nanoparticles.
10. The process of claim 1, wherein the primary SERS nanoparticles
have an average particle diameter of less than 200 nanometers.
11. The process of claim 1, wherein the primary SERS nanoparticles
have an average particle diameter at 50 to 100 nanometers.
12. A process for amplifying a Raman signal associated with primary
SERS nanoparticles, comprising: binding the primary SERS
nanoparticles to a target organism, wherein the primary SERS
nanoparticles comprise a reporter molecule for identifying the
primary SERS nanoparticles; and binding secondary SERS
nanoparticles to the primary SERS nanoparticles, wherein said
binding secondary SERS nanoparticles is effective to amplify the
Raman signal relative to primary SERS nanoparticles without bound
secondary SERS nanoparticles.
13. The process of claim 12, wherein the secondary SERS
nanoparticles contain a different reporter molecule than the
reporter molecule of the primary SERS nanoparticles.
14. The process of claim 12, wherein the secondary SERS
nanoparticles contain the reporter molecule of the primary SERS
nanoparticles.
15. The process of claim 12, wherein the primary and secondary SERS
nanoparticles are formed from a metal colloid.
16. The process of claim 15, wherein the metal colloid contains a
metal selected from a group consisting of gold, silver, copper,
sodium, aluminum, chromium, and alloys containing at least one of
these metals.
17. The process of claim 12, wherein the secondary SERS
nanoparticles comprise reporter molecules that exhibit
superparamagnetism.
18. The process of claim 12, wherein the primary SERS nanoparticles
have a negative charge and the secondary SERS nanoparticles have a
positive charge.
20. A process for amplifying a Raman signal associated with primary
SERS nanoparticles, comprising: depositing secondary SERS
nanoparticles from a solution onto immobilized primary SERS
nanoparticles; and aggregating the secondary SERS nanoparticles
about the immobilized SERS nanoparticles.
21. The process of claim 20, wherein said depositing secondary SERS
nanoparticles comprises precipitating the secondary SERS
nanoparticles by drying the solution.
22. The process of claim 20, wherein said depositing secondary SERS
nanoparticles comprises destabilizing the secondary SERS
nanoparticles solution by addition of a salt or other destabilizing
material and forcing the solution containing the secondary SERS
nanoparticles to aggregate about immobilized primary SERS
particles.
23. The process of claim 22, wherein said destabilizing secondary
SERS nanoparticles comprises precipitating a portion of the
secondary SERS nanoparticles and forming aggregates about the
immobilized primary SERS nanoparticles.
24. The process of claim 20, wherein the primary SERS nanoparticles
have a positive charge and the secondary SERS nanoparticles have a
negative charge
Description
BACKGROUND OF THE INVENTION
[0001] The disclosure generally relates to methods for amplifying
the Raman signal of surface enhanced Raman scattering (SERS)
nanoparticles.
[0002] Surface enhanced Raman scattering allows for the detection
of molecules attached to the surface of a single metallic
nanoparticle, typically a gold or silver nanoparticle. Existing
SERS nanoparticles, also referred to as nanotags, generally include
the metallic nanoparticle having a reporter molecule in close
proximity thereto (typically less than 50 angstroms), which
produces a strong Raman signal due to a surface enhanced effect.
Bringing reporter molecules in close proximity to the metal
surfaces is typically achieved by adsorption of the Raman-active
molecule onto suitably roughened metal nanoparticles, e.g., gold,
silver, copper, or other free electron metals. The characteristic
signal of the reporter molecule is used to determine the presence
and amount of the SERS nanoparticles. Consequently, SERS
nanoparticles have utility as spectroscopic and optical tags and
are often used in assays.
[0003] SERS nanoparticles are somewhat versatile and can be
functionalized with biological molecules (e.g., antibodies, DNA,
and the like) so that they specifically bind to one kind of target
(e.g., specific types of bacteria, viruses, spores, proteins, DNA,
and the like). For example, SERS nanoparticles can be used in
immunoassays when conjugated to an antibody against a target of
interest. If the target of interest is immobilized on a solid
support, then the interaction between a single target and a single
nanoparticle-bound antibody could be detected by searching the
unique Raman spectrum for the Raman-active reporter molecule.
Furthermore, because a single Raman spectrum (from 100 to 3500
cm.sup.-1) can detect many different Raman-active molecules, SERS
nanoparticles can often be used in multiplexed assay formats.
[0004] SERS is believed to occur primarily as a result of surface
plasmon resonances in the metal nanoparticle that enhance the local
intensity of the light, and formation and subsequent transitions of
charge-transfer complexes between the metal surface and the
Raman-active reporter molecule.
[0005] Protocols for producing SERS nanoparticles from colloidal
solutions of metallic nanoparticles present formidable practical
problems. For example, metal nanoparticles are exceedingly
sensitive to aggregation in aqueous solution; once aggregated,
re-dispersion is generally impossible. In addition, the chemical
compositions of some Raman-active reporter molecules are
incompatible with the chemistries used to attach other molecules
(such as proteins) to the metal nanoparticles. This restricts the
choices of Raman-active reporter molecules, attachment chemistries,
and other molecules to be attached to the metal nanoparticle.
[0006] By design, the Raman spectroscopic signal from a SERS
nanotags is dominated by the surface-enhanced Raman spectrum of the
attached reporter molecule. Other attached moieties, such as the
recognition element (e.g., antibody), or encapsulating shells
(e.g., glass), do not contribute to the observed Raman spectrum
because they are either Raman inactive, their Raman signal is not
surface-enhanced due to the particular bonding geometry, their
distance from the metal surface is too great, or other reasons.
[0007] Current processes for making the SERS nanoparticles are
numerous. One method as described in U.S. Pat. No. 6,514,767 to
Natan generally follows a synthetic pathway as outlined in the
schematic provided in prior art FIG. 1. The synthetic pathway
generally starts with a colloidal solution, e.g., HAuCl.sub.4
(i.e., gold chloride) colloidal solution, and a reducing agent that
results in the precipitation of gold nanoparticles having average
diameters of about 60 nm. The reducing agent is composed of a
single reductant, typically a citrate salt, e.g., sodium citrate,
to reduce the gold and form a stable colloid. The resulting colloid
is generally red in color and exhibits an absorption peak
(.lamda..sub.max) at about 530 nm. An amino-based silane is then
added to form vitreophilic surfaces capable of accepting the
desired tag or reporter molecules. Next, a silicate is added, which
polymerizes onto the "tagged" gold nanoparticle surface. The
thickness of the silicate layer is typically on the order of a few
nanometers. A thicker shell can be formed if desired using
tetraethylorthosilicate (TEOS). During or after this step, the
glass-coated nanoparticle can also be functionalized such as with
3-mercaptopropyltrimethoxysilane (MPTMS) or
3-aminopropyltrimethoxysilane (APTMS) to form SERS nanoparticles
with corresponding end groups having sulfhydryl or amino
functionalization. Optionally, a second silane-coupling agent can
be used depending on the polarity of the solvent in which the
particles are to be dispersed. In this manner, the nanoparticles
can be dispersed in a low polarity solvent if desired for the
particular application. Target molecules with the appropriate
linker chemistry are reacted with the end groups to provide the
tagged SERS nanoparticles. For example, antibody conjugated SERS
nanoparticles can be formed.
[0008] Other methods for producing SERS-active nanotags provide
different particle architectures. For example, Nie and Doering as
described in PCT Application No. WO 2005/062741 used organic dyes
adsorbed to a metallic core, and also encapsulated the resulting
particle with a glass shell. In U.S. Patent Publication No.
US20050089901 A1 to Porter, a tag is built from a metal
nanoparticle core, and in this case, the Raman-active molecule is
specifically chosen to have a reactive end that binds to the metal
nanoparticle surface and another part that acts as a linker to the
biological attachment part, so that the overall SERS nanotags does
not have a glass shell. In US Published Patent Application No.
2005/0158877 A1 to Wang et al., analyte analogues are first
attached to a metallic particle surface. Then the metallic
colloidal solution is mixed with an antibody solution. Each
antibody molecule will bind with two analyte analogue molecules,
thus causing the metallic particles to aggregate and form a cluster
structure for SERS signal amplification. In the presence of
analyte, the antibody molecule reacts with the analyte molecule and
the formation of the cluster structure is inhibited, which results
in a decrease of the Raman signal. Thus the presence and amount of
the analyte can be inferred from the intensity variation of the
Raman signals. In each instance, each SERS particle has both the
Raman dye and the antibody.
[0009] During the preparation of SERS nanotags, starting with a
colloidal solution of metallic nanoparticles, it is often observed
that the Raman signal of the adsorbed reporter molecule is
substantially enhanced when the metallic nanoparticles begin to
aggregate due to addition of an excess amount of the reporter
molecule, or an excess of another adsorbate along with the reporter
molecule. However, gross aggregation prevents further processing of
the SERS nanotags, since they begin to form precipitates that are
not re-dispersible, and are often not amenable to further
modification such as antibody attachment. Thus, protocols for
preparation of SERS nanotags are designed to eliminate or minimize
aggregation.
[0010] Regardless of the method used for producing the SERS active
nanoparticles, there remains a need in the art for amplification of
the Raman signal to improve the detection limits especially with
regard to the SERS nanoparticles.
SUMMARY OF THE INVENTION
[0011] Processes for amplifying the Raman signal of primary SERS
nanoparticles are described herein. In one embodiment, the process
for amplifying a Raman signal associated with primary SERS
nanoparticles, wherein the SERS nanoparticles contain a metallic
core comprises aggregating secondary SERS nanoparticles about the
primary SERS nanoparticles after a binding event between the
primary SERS nanotags and a target analyte, wherein the secondary
SERS nanoparticles contain a different reporter molecule than the
primary SERS nanoparticles or are free from the reporter molecule.
In this case, the Raman signal originating from a primary SERS
nanoparticle is amplified due to a coupling effect (plasmon effect)
between the attached primary SERS nanoparticle and the secondary
nanoparticles that are caused to aggregate around the primary SERS
nanoparticle.
[0012] In another embodiment, the process for amplifying a Raman
signal associated with primary SERS nanoparticles comprises binding
the primary SERS nanoparticles to a target organism, wherein the
primary SERS nanoparticles comprise a reporter molecule for
identifying the primary SERS nanoparticles; and binding secondary
SERS nanoparticles to the primary SERS nanoparticles, wherein
binding the secondary SERS nanoparticles are effective to amplify
the Raman signal relative to primary SERS nanoparticles without the
bound secondary SERS nanoparticles. In this case, the Raman signal
is amplified due to the additive signals arising from the attached
primary SERS nanotags, plus the Raman signal from the secondary
SERS nanotags that have been enabled to bind to the primary SERS
nanotags.
[0013] In yet another embodiment, a process for amplifying a Raman
signal associated with primary SERS nanoparticles comprises
depositing secondary SERS nanoparticles from a solution onto
immobilized primary SERS nanoparticles; and aggregating the
secondary SERS nanoparticles about the immobilized SERS
nanoparticles.
[0014] The above described and other features are exemplified by
the following figures and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a prior art schematic illustrating a synthetic
pathway for producing SERS nanoparticles.
[0016] FIG. 2 schematically illustrates aggregation of secondary
SERS tags around a primary SERS nanotag after the primary SERS
nanotag attached to a target molecule via a bio-recognition
event.
[0017] FIG. 3 schematically illustrates binding of secondary SERS
particles to a primary SERS particle after the primary SERS
particle has been bound to the target analyte.
[0018] FIG. 4 graphically illustrates overlayed Raman spectra
showing the effect of aggregating additional gold colloid onto SERS
nanotags [functionalized with a reporter molecule
1,2-bis(4-pyridyl)ethylene (BPE)] that have been immobilized by
depositing them onto a filter paper.
[0019] FIG. 5 graphically illustrates overlayed Raman spectra from
immobilized BPE-labeled SERS nanotags before and after they were
treated with additional unlabeled colloid, under conditions where
the unlabeled colloid aggregates around the primary SERS
nanotags.
[0020] FIG. 6 graphically illustrates overlayed Raman spectra
showing the signal of assays performed with primary SERS particles
then with primary and secondary SERS particles.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0021] The present disclosure is generally directed to amplifying
the Raman signal of SERS nanoparticles. In one embodiment, one or
more secondary nanoparticles ("amplifier" particles) are brought
into close proximity (a few nanometers or less) to the primary SERS
nanoparticle, thereby essentially forming a pseudo-like aggregate
of nanoparticles clustered around the SERS nanoparticle. The SERS
nanoparticles that have the Raman signal amplified by secondary
nanoparticles are also referred to herein as the primary SERS
nanoparticles.
[0022] As noted in the background section, under normal
circumstances aggregation is not desirable since it prevents the
SERS nanoparticles from being isolated and purified as well as
provides limits to their solubility and mobility. In this case, the
aggregate is formed after the reporter molecule of the SERS
nanoparticle has been formed or after the SERS nanoparticles have
been used in an assay. The close proximity of the secondary
nanoparticles to the primary SERS nanoparticles provides for
further amplification of its Raman signal. These secondary
"amplifier" nanoparticles can be forced to aggregate around the
primary SERS nanoparticle by specific interactions such as by
chemical or biological binding mechanisms, by electrostatic
attraction or by non-specific deposition methods such as by
evaporative drying of a nanoparticle solution containing the SERS
nanoparticle and secondary amplifier nanoparticles. The one or more
secondary nanoparticles are similar in geometry and composition to
the SERS nanoparticle with the exception that the secondary
nanoparticles do not contain the same reporter molecule. In other
embodiments the secondary nanoparticles contain a reporter molecule
that is different from the one absorbed onto the primary SERS
nanoparticles. As such, the secondary nanoparticles do not have to
be Raman active but can be merely a stable metal colloid.
[0023] Specific interactions between the secondary nanoparticles
and the primary SERS nanoparticles can form a sub-monolayer, a
monolayer or a multilayer coating on the SERS nanoparticle.
Coatings such as glass or a polymer can also be used to cover the
SERS nanoparticles, thereby preventing secondary amplifier
molecules from directly contacting SERS nanoparticles and allowing
for separation of the amplifier molecules from the SERS
nanoparticles by a distance dictated by coating thickness (glass or
polymer) while still allowing for sufficient amplification. Initial
results with glass coated SERS nanoparticles (a glass coating of 5
to 50 nanometers) suggest that the metallic cores need not be
touching, but can be separated by the distance dictated by the
coating with the amplification effect still being observed. In one
embodiment, the distance defined by the coating is less than 5
nanometers.
[0024] In another embodiment, assays employing the primary SERS
nanoparticle include the use of secondary SERS nanoparticles (i.e.,
tags), which are targeted to moieties on the primary SERS
nanoparticle. In this embodiment, the secondary nanoparticles are
SERS active. This process results in increasing the number of SERS
active particles bound to the target, either directly, or via their
association with the primary particle attached to the target,
thereby increasing signal and assay sensitivity. Specifically, the
use of the secondary SERS nanoparticles may increase the number of
tags associated with each targeted organism for assay and therefore
the sensitivity of the assays. Advantageously, the secondary SERS
nanoparticles can also include tags with other features as may be
desirable for different applications, e.g., superparamagnetism.
[0025] By way of example, a sample material that may contain a
target organism or molecule is contacted with the primary SERS
nanoparticles that have been functionalized with, for example,
antibodies, against the target organism. If the target organism is
present, the primary SERS nanoparticles will bind to the target
organism due to target specific antibodies present of the target
organism surface. Unbound primary SERS nanoparticles are then
removed in a washing step. Quantities of secondary SERS
nanoparticles functionalized with antibodies that bind to the
primary SERS nanoparticles are then added. The secondary SERS
nanoparticles are Raman active but can also include tags with other
features such as superparamagnetism. The unbound secondary SERS
nanoparticles are then removed by washing. The above noted process
results in an increased number of SERS active particles bound to
the target, thereby increasing the signal and assay sensitivity.
The sample containing the target molecules or organisms can then be
analyzed for Raman intensity using a laser and Raman spectrometer
in a manner well known in the art.
[0026] The secondary SERS nanoparticles can be exclusively or
predominantly SERS active and these can have the same or different
Raman signature as the primary SERS nanoparticles. The use of two
different Raman signatures can enhance assay specificity by
reducing the number of false positives. Moreover, as previously
noted, the secondary SERS nanoparticles can include
superparamagnetic particles that can be used to immobilize the
target complex to reduce background signal.
[0027] As a result of the amplification provided by the secondary
nanoparticles in either of the processes disclosed above, increased
signal intensity, lower limits of detection, and a reduced false
positive rate during detection can be obtained. The methods
disclosed herein can be used for individually tagging a molecule,
cell, bead or solid support to isolate the signal or may be used
for multiplexing. Moreover, the amplification process is not
intended to be limited to any particular method for producing the
SERS nanoparticles, which may or may not include a glass
coating.
[0028] In accordance with some embodiments, the metallic cores of
the nanoparticles are preferably formed from a metal selected from
the group consisting of Au, Ag, Cu, Na, Al, Cr as well as alloys.
The metal nanoparticles are generally less than 200 nanometers (nm)
in diameter. The theory associated with amplification of the Raman
signal from SERS nanoparticles (SERS=surface-enhanced Raman
scattering) using aggregation-inducing techniques begins with the
understanding that existing SERS nanoparticles consist of a
nanoparticles having a metallic core to which is adsorbed a
reporter molecule, wherein the reporter molecule gives a strong
Raman signal due to the surface-enhanced Raman effect. It is
believed that the signal from the reporter molecules on the SERS
nanoparticles is further amplified when it is sandwiched between
the SERS nanoparticles, or between SERS nanoparticles and a planar
gold surface. The characteristic signal of the reporter molecule is
used to determine the presence and amount of SERS nanoparticles,
which can be used in detection schemes (bioassays) of various
formats. For example, the SERS nanoparticles can be functionalized
with biological molecules as previously noted (e.g., antibodies,
DNA, etc) so that they specifically bind to one kind of target
organism (e.g., specific types of bacteria, viruses, spores,
proteins, DNA, and the like).
[0029] The reporter molecule selected for the SERS nanoparticles is
not intended to be limited; the selection of which are generally
dependent on the particular application. Commonly employed reporter
molecules include, but are not limited to, dyes,
1,2-bis(4-pyridyl)ethylene, 4,4'-bipyridyl, 2-qunolinethiol, and
4-mercaptopyridine, and the like. The selection of a reporter
molecule for a particular application is well within the skill of
those in the art in view of this disclosure.
[0030] As noted above, in one embodiment as shown more clearly in
FIG. 2, the one or more of the secondary nanoparticles 12 are
brought into close proximity to the primary SERS nanoparticles 10
after bio-recognition of the primary SERS nanoparticles to a target
molecule 14, essentially forming a pseudo-like aggregate of
nanoparticles clustered around the SERS nanoparticles. Aggregation
about the primary SERS nanoparticles can be caused by electrostatic
attraction, chemical or biological binding as previously discussed.
In this manner, signal amplification can be differentiated from
brighter tags. Therefore, one of the advantages provided by
embodiments herein includes the use of an aqueous solution and
stable reagents to provide the aggregation effect. In one
embodiment, SERS-active nanoparticles were modified with an amine
in an attempt to create particles with a positive charge to form
the secondary SERS nanoparticles. These can be combined with plain
or glass-coated SERS-active particles, which have a negative charge
such as those produced in accordance with FIG. 1. Aggregation
results after the primary SERS nanoparticles have been attached to
the target organism resulting in signal amplification upon
aggregation of secondary nanoparticles. It should also be apparent
that the electrostatic attraction could vary. For example, the
primary SERS nanoparticles could have a positive charge and the
secondary nanoparticles a negative charge.
[0031] FIG. 3 schematically illustrates binding of secondary SERS
particles to a primary SERS particle after the primary particle
bound to a target analyte.
[0032] Increasing the Raman signal of individual SERS nanoparticles
will improve the detection limit. In a bioassay, the signal from a
single binding event (one SERS nanoparticle) can be amplified by
the methods disclosed herein in a second step when secondary
particles are introduced after the binding event. The Raman signal
associated with the single SERS nanoparticles can then be detected
because of the amplification. Existing methods for coupling
antibodies to surfaces or to other molecules are well known with
commercial reagents and protocols readily available to those in the
art.
[0033] The following examples are presented for illustrative
purposes only, and are not intended to limit the scope of the
invention.
EXAMPLE 1
[0034] In this example, 60 nanometer gold colloid solutions were
prepared. A 2 liter, 3-necked round-bottom flask with stirring
provided by an electric overhead stirrer (Arrow Engineering
#JR4000) with a Teflon.RTM. paddle was immersed in an ice bath.
Using freshly prepared and pre-cooled reagent solutions, the gold
colloid solution was prepared as follows. The glassware was first
cleaned with aqua regia (3 parts HCl:1 part HNO.sub.3) and between
batches of colloid and rinsed with Milli-Q distilled deionized
water. A Raman spectrometer consisting of the components from
General Electric Company's StreetLab.RTM., and controlled by Ocean
Optics software was utilized.
[0035] A stock solution of 1% HAuCl.sub.43H.sub.2O was prepared by
dissolving the solid in Milli-Q distilled deionized water. For
example, 2 grams HAuCl.sub.43H.sub.2O in about 10 milliliter water,
then transfer quantitatively into a 200 milliliter volumetric flask
and dilute. The solution was filtered through a 0.2 micron
cellulose acetate filter and stored in a dark environment prior to
use. A stock solution of 1 Normal NaOH was prepared from a Baker
Dilut-it cartridge. Dilute a portion to provide 0.01 Normal NaOH
for reagent preparations (500 milliliters diluted to 50
milliliters). The remainder of the ingredients were prepared the
day of the synthesis and stored on ice.
[0036] A 32% (w/v) trisodium citrate dihydrate solution in 0.01N
Normal NaOH was prepared by weighing 3.2 grams (g) trisodium
citrate dihydrate in a 10-milliliter tube top which 0.01 Normal
NaOH was added to make 10 milliliters. Complete dissolution
required several minutes of mechanical agitation.
[0037] A 1.6 M hydroxylamine hydrochloride solution was prepared by
weighing 1.116 g hydroxylamine hydrochloride in 10 milliliters of
Milli-Q water.
[0038] Into the assembled flask and stirring assembly, 24
milliliters (ml) of the 1% HAuCl.sub.4 stock solution was added to
976 milliliter of (ice cold) Milli-Q water to form 1 liter of
0.024% HAuCl.sub.4. To this solution, 1.00 ml of the hydroxylamine
solution was added with continued stirring. Exactly 20 minutes
after the hydroxylamine was added to the gold solution, 1 ml of a
reductant solution was added. The reductant solution was prepared
by mixing 1.0 ml of the 32% citrate solution, 525 microliters of
0.01 Normal (N) NaOH, and 75 microliters of the 0.0004% sodium
borohydride. Within seconds, the solution changed rapidly in color
from yellow to deep purple to red. The solution is stirred for
about 1 minute after which the stirring assembly is dismantled and
the colloid transferred to a flask at room temperature. An aliquot
of the resulting colloid is then diluted with an equal part water
before characterization by UV-Visible spectroscopy. The resulting
colloids had an absorbance of 1.3-1.5 and .lamda..sub.max=528-534
nanometers.
EXAMPLE 2
[0039] In this example, 2 microliters of the gold colloid solution
of Example 1 was deposited onto filter paper. To this, a 1
millimole (mM) solution in ethanol of 1,2-bis(4-pyridyl)ethylene
(BPE) was deposited. As noted above, BPE is a reporter molecule. To
this 6 microliters (.mu.l) of plain colloid was added. The Raman
spectrum was periodically measured and is graphically shown in FIG.
4. From the spectra, it was observed that the signal intensity
corresponding to the reporter molecule dramatically increased upon
addition of the plain colloid, which is indicative of aggregate
formation.
EXAMPLE 3
[0040] In this example, the colloid and BPE were mixed prior to
deposition of 2 .mu.l onto filter media followed by drying. The
filter media was a porous hydrophilic acrylic copolymer
commercially available under the trade name Versapor from the Pall
Corporation. A colloid solution was then added to the colloid/BPE
mixture. Raman spectra was periodically obtained, the results of
which are provided in FIG. 5. Signal amplification was observed
upon addition of the colloid.
[0041] The primary SERS nanotags were prepared in situ by first
depositing 2 microliters of gold colloid (60 nm diameter,
2.3.times.10.sup.10 particles/mL) onto the filter paper. One drop
of a solution of pre-formed gold nanoparticle aggregates (2 mL
colloid plus 1 drop 500 M NaCl) was deposited onto the spot
containing the immobilized primary SERS nanotags. The presence of
the unlabeled gold nanoparticle aggregates around the immobilized
BPE-labeled primary SERS nanotags causes an amplification of its
Raman signal.
EXAMPLE 4
[0042] In this example, a solution of primary SERS tags with BPE as
the Raman reporter molecule was mixed with a target analyte. The
primary SERS tags bound to the target analyte and the excess were
washed away. The signal intensity from this assay was observed and
the resulting data is shown as the less intense traces in FIG. 6.
Secondary SERS tags (also with BPE as the Raman reporter molecule)
were then introduced into the system and specifically bound to the
primary tags via an antibody interaction and the excess tags were
washed away. The signal intensity from this assay was observed and
the resulting data is shown in FIG. 6. The traces depicting the
lower signal indicate the assay performed with primary tags alone
whereas the traces depicting higher signal indicate the assay
results when performed with the primary and secondary tags after
the binding of the secondary tags. Clearly, it can be seen that
signal amplification occurred upon the binding of secondary SERS
tags to the primary SERS tags already bound to the target.
[0043] This written description uses examples to disclose the
invention, including the best mode, and also to enable any person
skilled in the art to make and use the invention. The patentable
scope of the invention is defined by the claims, and may include
other examples that occur to those skilled in the art. Such other
examples are intended to be within the scope of the claims if they
have structural elements that do not differ from the literal
language of the claims, or if they include equivalent structural
elements with insubstantial differences from the literal languages
of the claims.
* * * * *